Abstract
A assay was developed to detect the level of contaminating male fertile rapeseeds within a population of male sterile Ogu-INRA seeds, based on PCR amplification of DNA and fluorescence technology. A molecular beacon was constructed to recognize a specific sequence of the male fertile plant. However, this sequence was also present in the male sterile plant at a very low concentration because of the recombinant structure of the Ogu-INRA mitochondrial genome. Conditions for quantitative PCR were found to take into account this biological data in order to develop an effective test. A 1% contamination can be detected with the naked eye with an ultraviolet light table or by use of a spectrofluorometer. The sensitivity of this method is dependent upon the molecular beacon concentration.
Similar content being viewed by others
References
Bellaoui M., Pelletier G. and Budar F. 1998. Identification of a rapeseed locus which determines the presence of a substoichiometric mitochondrial molecule in cybrid cytotype. In: Moller I.M., Gardestrom P. and Glaser E. (eds), Plant Mitochondria: from Gene to Function. pp. 63–68.
Bonhomme S., Budar M., Férault F. and Pelletier G. 1991. A 2.5 kb NcoI fragment of Ogura radish mitochondrial DNA is correlated with cytoplasmic male sterility in Brassica cybrids. Curr. Genet. 19: 121–127.
Bonhomme S., Budar F., Lancelin D., Small I., Defrance M.C. and Pelletier G. 1992. Sequence and transcript analysis of the Nco 2.5 Ogura-specific fragment correlated with cytoplasmic malesterility in Brassica cybrids. Mol. Gen. Genet. 235: 340–348.
Fauron C., Casper M., Gao Y. and Moore B. 1995. The maize mitochondrial genome: dynamic, yet functional. Trends Genet. 11: 228–235, Review.
Kota R., Holton T.A. and Henry R.J. 1999. Detection in crops plants using molecular beacon assays. Plant Molecular Biology Reporter 17: 363–370.
Leone G., Van Schijndel H., Van Gemen B., Kramer F.R. and Schoen C.D. 1998. Molecular beacon probes combined with the amplification of NASBA enable homogeneous, real time detection of RNA. Nucleic Acids Res. 26: 2150–2155.
Piatek A.S., Tyagi S., Pol A.C., Telenti A., Miller L.P., Kramer F.R. et al. 1998. Molecular Beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nature Biotechnol. 16: 359–363.
Stryer L. 1978. Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47: 819–846.
Tinchant C., Defrance M.C. and Budar F. 1997. PCR primers for the estimation of contamination in rape seed lots bearing malesterility inducing OGU-INRA cytoplasm by seeds with normal cytoplasm. Plant Breeding 116: 390–392.
Tyagyi S. and Kramer F.R. 1996. Molecular Beacons: Probes that fluoresce upon hybridization. Nature Biotechnol. 14: 303–308.
Tyagyi S., Bratu D.P. and Kramer F.R. 1998. Multicolor molecular beacons for allele discrimination. Nature Biotechnol. 16: 49–53.
Vedel F., Mathieu C., Chétrit P., Pelletier G. and Primard C. 1987. Mitochondrial DNA variation in cytoplasmic male sterile somatic hybrids of Brassica napus. Plant Physio. Biochem. 25: 249–257.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Rousseau, S., Brunel, D. Use of molecular beacon for the detection of fertile male plants in batches of sterile male rapeseed. Molecular Breeding 12, 91–96 (2003). https://doi.org/10.1023/A:1026013114878
Issue Date:
DOI: https://doi.org/10.1023/A:1026013114878